Inositol 1,4,5-trisphosphate receptor expression in cardiac myocytes.

Calcium release from intracellular stores is the signal generated by numerous regulatory pathways including those mediated by hormones, neurotransmitters and electrical activation of muscle. Recently two forms of intracellular calcium release channels (CRCs) have been identified. One, the inositol 1,4,5-trisphosphate receptors (IP3Rs) mediate IP3-induced Ca2+ release and are believed to be present on the ER of most cell types. A second form, the ryanodine receptors (RYRs) of the sarcoplasmic reticulum, have evolved specialized functions relevant to muscle contraction and are the major CRCs found in striated muscles. Though structurally related, IP3Rs and RYRs have distinct physiologic and pharmacologic profiles. In the heart, where the dominant mechanism of intracellular calcium release during excitation-contraction coupling is Ca(2+)-induced Ca2+ release via the RYR, a role for IP3-mediated Ca2+ release has also been proposed. It has been assumed that IP3Rs are expressed in the heart as in most other tissues, however, it has not been possible to state whether cardiac IP3Rs were present in cardiac myocytes (which already express abundant amounts of RYR) or only in non-muscle cells within the heart. This lack of information regarding the expression and structure of an IP3R within cardiac myocytes has hampered the elucidation of the significance of IP3 signaling in the heart. In the present study we have used combined in situ hybridization to IP3R mRNA and immunocytochemistry to demonstrate that, in addition to the RYR, an IP3R is also expressed in rat cardiac myocytes. Immunoreactivity and RNAse protection have shown that the IP3R expressed in cardiac myocytes is structurally similar to the IP3R in brain and vascular smooth muscle. Within cardiac myocytes, IP3R mRNA levels were approximately 50-fold lower than that of the cardiac RYR mRNA. Identification of an IP3R in cardiac myocytes provides the basis for future studies designed to elucidate its functional role both as a mediator of pharmacologic and hormonal influences on the heart, and in terms of its possible interaction with the RYR during excitation-contraction coupling in the heart.

Differential regulation of two types of intracellular calcium release channels during end-stage heart failure.

The molecular basis of human heart failure is unknown. Alterations in calcium homeostasis have been observed in failing human heart muscles. Intracellular calcium-release channels regulate the calcium flux required for muscle contraction. Two forms of intracellular calcium-release channels are expressed in the heart: the ryanodine receptor (RyR) and the inositol 1,4,5-trisphosphate receptor (IP3R). In the present study we showed that these two cardiac intracellular calcium release channels were regulated in opposite directions in failing human hearts. In the left ventricle, RyR mRNA levels were decreased by 31% (P < 0.025) whereas IP3R mRNA levels were increased by 123% (P < 0.005). In situ hybridization localized both RyR and IP3R mRNAs to human cardiac myocytes. The relative amounts of IP3 binding sites increased approximately 40% compared with ryanodine binding sites in the failing heart. RyR down-regulation could contribute to impaired contractility; IP3R up regulation may be a compensatory response providing an alternative pathway for mobilizing intracellular calcium release, possibly contributing to the increased diastolic tone associated with heart failure and the hypertrophic response of failing myocardium.

SM-20 is a novel growth factor-responsive gene regulated during skeletal muscle development and differentiation.

SM-20 is a novel, evolutionarily conserved "early response" gene originally cloned from a rat aortic smooth muscle cell (SMC) cDNA library. SM-20 encodes a cytoplasmic protein, which is induced by platelet-derived growth factor and angiotensin II in cultured SMC and is upregulated in intimal SMC of atherosclerotic plaques and injured arteries. We have now examined SM-20 expression during differentiation of cultured skeletal myoblasts and during skeletal myogenesis in vivo. Low levels of SM-20 mRNA and protein were expressed in proliferating mouse C2C12 myoblasts. Differentiation by serum withdrawal was associated with a marked induction of SM-20 mRNA and the expression of high levels of SM-20 antigen in myotubes. The induction was partially inhibited by blocking differentiation with bFGF or TGFbeta. Similar results were obtained with the nonfusing mouse C25 myoblast line, suggesting that SM-20 upregulation is a consequence of biochemical differentiation and is fusion independent. During mouse embryogenesis, SM-20 was first observed at 8.5E in the dermomyotomal cells of the rostral somites. SM-20 expression progressed in a rostral to caudal pattern, with highest levels seen in the muscle primordia and mature muscles. SM-20 thus represents a novel intracellular protein that is regulated during skeletal muscle differentiation and development.

Transient and chronic neonatal denervation of murine muscle: a procedure to modify the phenotypic expression of muscular dystrophy.

The extensor digitorum longus muscles of 14-d-old normal (129 ReJ ++) and dystrophic (129 ReJ dy/dy) mice were denervated by cutting the sciatic nerve. One denervation protocol was designed to inhibit reinnervation of the shank muscles, the other to promote reinnervation. Chronically denervated muscles (muscles that remained denervated for 100 d after nerve section) exhibited marked atrophy, but the number of myofibers in these muscles (1066 +/- 46 and 931 +/- 62 for the denervated normal and dystrophic muscles, respectively) was similar to the number of myofibers found in age-matched, unoperated normal muscles [922 +/- 28 (Ontell et al., 1984)] and was significantly greater than the number of myofibers found in age-matched dystrophic muscles [547 +/- 45 (Ontell et al., 1984)]. Similar effects on myofiber number were obtained when denervated muscles were allowed to reinnervate. Reinnervation of both normal and dystrophic muscles mitigated the marked atrophy that characterized chronically denervated muscles. The dystrophic reinnervated muscles appeared "healthier" than age-matched, unoperated dystrophic muscles, having 70% more myofibers, less myofiber diameter variability, substantially less connective tissue infiltration, and a greater amount of contractile tissue at their widest girths. The present study demonstrated that it is possible to alter the phenotypic expression of the histopathological changes associated with murine dystrophy, in dystrophic myofibers that are formed during fetal development, by subjecting the muscle to neonatal denervation.

Effect of neonatal denervation-reinnervation on the functional capacity of a 129ReJ dy/dy murine dystrophic muscle.

The sciatic nerves of 14-day-old 129 ReJ normal (++) and dystrophic (dy/dy) mice were transected in the mid-thigh region. The cut ends of the nerves were approximated to facilitate regeneration. One hundred days after denervation, contractile properties of denervated-reinnervated, normal and dystrophic extensor digitorum longus (EDL) muscles were compared to age-matched normal and dystrophic muscles. In dystrophic muscle, in vitro twitch and tetanic tensions were reduced, compared to those of normal muscle. The denervation-reinnervation procedure resulted in an increase in these parameters as compared to unoperated dy muscle. These data correlated with increases in total myofiber cross-sectional areas. Twitch contraction time was not significantly affected by the dystrophic condition or by the denervation-reinnervation protocol. Whereas dystrophic muscle had a longer half-relaxation time than normal muscle, denervation-reinnervation of the dystrophic EDL resulted in a significantly faster half-relaxation time. While fatigue resistance was greater in dystrophic muscles than in normal muscle, there was a significant decrease in fatigue resistance in the denervated-reinnervated dystrophic muscle. Transient neonatal denervation results in modification of both the morphological and physiological characteristics of murine dystrophy.

Inositol 1,4,5-trisphosphate receptor in skeletal muscle: differential expression in myofibres.

The role of inositol 1,4,5-trisphosphate as a second messenger in signal transduction has been well established in many cell types. However, conflicting reports have led to a controversy regarding the role, if any, of inositol 1,4,5-trisphosphate signalling in skeletal muscle. Indeed, expression of the inositol 1,4,5-trisphosphate receptor has not previously been demonstrated in skeletal muscle. In the present study we used in situ hybridization, immunohistochemistry, and [3H]-inositol 1,4,5-trisphosphate binding to demonstrate that rat skeletal muscle fibres contain inositol 1,4,5-trisphosphate receptors. RNAse protection and partial sequencing suggested that the inositol 1,4,5-trisphosphate receptors expressed in skeletal muscle was most similar to the non-neuronal form of the type 1 inositol 1,4,5-trisphosphate receptor. While in situ hybridization showed inositol 1,4,5-trisphosphate receptor mRNA in all types of skeletal myofibres, immunodetectable inositol 1,4,5-trisphosphate receptor protein and specific [3H]-inositol 1,4,5-trisphosphate binding sites were preferentially expressed in slow oxidative (type I) and fast oxidative-glycolytic (type IIA) fibres, but not in fast glycolytic (type IIB) fibres. These findings indicate that an inositol 1,4,5-trisphosphate receptor is preferentially expressed in oxidative fibres of skeletal muscle.

Sexually dimorphic expression of a laryngeal-specific, androgen-regulated myosin heavy chain gene during Xenopus laevis development.

Masculinization of the larynx in Xenopus laevis frogs is essential for the performance of male courtship song. During postmetamorphic (PM) development, the initially female-like phenotype of laryngeal muscle (slow and fast twitch fibers) is converted to the masculine form (entirely fast twitch) under the influence of androgenic steroids. To explore the molecular basis of androgen-directed masculinization, we have isolated cDNA clones encoding portions of a new Xenopus myosin heavy chain (MHC) gene. We have detected expression of this gene only in laryngeal muscle and specifically in males. All adult male laryngeal muscle fibers express the laryngeal myosin (LM). Adult female laryngeal muscle expresses LM only in some fibers. Expression of LM during PM development was examined using Northern blots and in situ hybridization. Males express higher levels of LM than females throughout PM development and attain adult levels by PM3. In females, LM expression peaks transiently at PM2. Treatment of juvenile female frogs with the androgen dihydrotestosterone masculinizes LM expression. Thus, LM appears to be a male-specific, testosterone-regulated MHC isoform in Xenopus laevis. The LM gene will permit analysis of androgen-directed sexual differentiation in this highly sexually dimorphic tissue.

Modification of the dystrophic phenotype after transient neonatal denervation: role of MHC isoforms.

While it recently has been demonstrated that it is possible to modify the phenotypic expression of murine dystrophy (dy/dy) (i.e., prevent myofiber loss) by subjecting the extensor digitorum longus (EDL) muscle of 14-day-old dy/dy mice to transient neonatal denervation (Moschella and Ontell, 1987), the mechanism responsible for this phenomenon has not been determined. Since it has been suggested that the effects of dystrophy vary according to fiber type, the fiber type frequency in 100-day-old normal (+/+) and dy/dy EDL muscles subjected to transient neonatal denervation has been determined by immunohistochemical analysis of their myosin heavy chain (MHC) composition. This frequency has been compared with that found in the EDL muscles of 14- and 100-day-old unoperated +/+ and dy/dy mice, in order to determine whether the reinnervation of transiently denervated neonatal muscle results in a preponderance of fibers of the type that might be spared dystrophic deterioration. In unoperated dy/dy muscle there is a progressive decrease in the frequency and in the absolute number of fibers that express MHC2B, with 100-day-old dy/dy muscles having approximately 32% of the number of myofibers fibers containing MHC2B as is found in age-matched +/+ muscles. The number of fibers containing the other fast isoforms (MHC2A and MHC2X) is similar in +/+ and dy/dy muscles at this age, indicating that fibers with MHC2B are most affected by the dystrophic process. Reinnervation following transient neonatal denervation of both the +/+ and the dy/dy EDL muscles results in a similar decrease (approximately 62%) in the number of myofibers containing MHC2B and an increase in myofibers containing the other fast MHC isoforms (MHC2A and MHC2X). The selective effect of dy/dy on fibers containing MHC2B and the sparing of myofibers in transiently denervated dy/dy muscle (which contains a reduced frequency of fibers containing MHC2B) are consistent with, although not direct proof of, the hypothesis that alterations in the fiber type may play a role in the failure of myofibers in transiently denervated dy/dy muscles to undergo dystrophic deterioration. Evidence is presented suggesting that neurons that supply myofibers containing MHC2B may be at a selective disadvantage in their ability to reinnervate neonatally denervated muscles.

Intracellular calcium release channel expression during embryogenesis.

The release of intracellular calcium (Ca2+) via either inositol 1,4, 5-trisphosphate receptors (IP3R) or ryanodine receptors (RyR) activates a wide variety of signaling pathways in virtually every type of cell. In the present study we demonstrate that at early stages of development IP3R mRNA and functional IP3-gated Ca2+ release channels are widely expressed in virtually all tissues in murine embryos. As organogenesis proceeds, more specialized RyR channels are expressed in many cell types and the triggering mechanisms for intracellular Ca2+ release become more diverse to include IP3-dependent and voltage-dependent and Ca2+-induced Ca2+ release. As development proceeds virtually all cell types continue to express IP3R channels but in excitable cells including skeletal and cardiac muscles the major Ca2+ release channels are RyRs. This developmental switch from predominantly IP3-mediated to both IP3-mediated and IP3-independent pathways for intracellular Ca2+ release is consistent with data showing that IP3R plays an important regulatory role in cellular proliferation and apoptosis, whereas RyR is required for other cellular functions including muscle contraction.

Stabilization of calcium release channel (ryanodine receptor) function by FK506-binding protein.

FK506-binding protein (FKBP12) was originally identified as the cytosolic receptor for the immunosuppressant drugs FK506 and rapamycin. The cellular function of FKBP12, a ubiquitously expressed 12,000-dalton proline isomerase, has been unknown. FKBP12 copurifies with the 565,000-dalton ryanodine receptor (RyR), four of which form intracellular Ca2+ release channels of the sarcoplasmic and endoplasmic reticula. By coexpressing the RyR and FKBP12 in insect cells, we have demonstrated that FKBP12 modulates channel gating by increasing channels with full conductance levels (by > 400%), decreasing open probability after caffeine activation (from 0.63 +/- 0.09 to 0.04 +/- 0.02), and increasing mean open time (from 4.4 +/- 0.6 ms to 75 +/- 41 ms). FK506 or rapamycin, inhibitors of FKBP12 isomerase activity, reverse these stabilizing effects. These results provide the first natural cellular function for FKBP12, and establish that the functional Ca2+ release channel complex includes FKBP12.